The expression of recombinant proteins for commercial production and biomedical research is a major undertaking in molecular biology. Recent efforts towards the development of “structural genomics,” in which protein structures for entire genomes are to be determined, create increased demand for simple, high-throughput cloning/expression systems (Walhout et al., 2000; Chandonia and Brenner, 2006; Blow, 2008).
While a variety of host organisms have been used to produce foreign proteins, the most commonly employed microorganism is the gram-negative bacterium, Escherichia coli. A common limitation of recombinant protein expression in E. coli is the toxicity of overexpressed proteins (Chen, 1994; Miroux and Walker, 1996).
Inducible Expression of Recombinant Proteins in E. coli Plasmid Vectors:
Host-vector systems that allow tightly regulated, inducible gene expression in E. coli are greatly preferred. Many different strains of E. coli and several DNA cloning vectors for protein expression have been developed (Bolivar et al., 1977; Yannisch-Perron et al., 1985; Denhardt and Colasanti, 1988; Godiska et al., 2005). The most widely used recombinant DNA cloning systems for regulated expression of proteins in E. coli rely upon transcription using bacteriophage T7 RNA polymerase (Studier and Moffatt, 1986; Rosenberg et al., 1987).
T7 Expression Systems:
T7 plasmid expression systems take advantage of (1) the specificity of binding of T7 bacteriophage RNA polymerase to its cognate DNA promoter (Rong et al., 1998) and (2) the high efficiency of T7 RNA polymerase-catalyzed transcription (Davanloo et al., 1984). In order to minimize synthesis of potentially toxic gene products, expression vectors are constructed so that transcription of target genes is under the control of T7 promoters on multi-copy plasmids (U.S. Pat. No. 4,952,496 to Studier et al., 1990; U.S. Pat. No. 5,693,489 to Studier et al., 1997). Transcription from T7 promoters occurs only in bacterial strains engineered to overproduce T7 RNA polymerase.
To minimize expression of deleterious gene products, cloning of DNA fragments under the control of a promoter for T7 RNA polymerase is performed in E. coli host strains that lack the gene for the T7 RNA polymerase, T7 gene 1 (T7gpl−). For downstream protein expression, the recombinant plasmid carrying the T7 promoter upstream of the target gene can be introduced into a second host strain which expresses T7 RNA polymerase. Although this dual-host strategy helps to limit unwanted expression of potentially toxic proteins, it also imposes a significant effort in terms of time and labor.
It is theoretically possible to design an improved plasmid host-vector system in which a single E. coli T7gpl+ host strain is used for both DNA transformation and subsequent expression of target genes. However, such a cloning/expression vector system is practical only if gene expression can be maintained under tight repression prior to induction, and if such repression can be overcome effectively upon induction.
State-of-the-Art T7 Expression Systems are Leaky:
E. coli systems used for T7-based expression place the target genes in recombinant plasmids under transcriptional control of T7 RNA polymerase (Dubendorff and Studier, 1991; U.S. Pat. No. 4,952,496 to Studier et al., 1990; U.S. Pat. No. 5,830,694 to Studier and Dubendorff, 1998). Commonly used strains such as E. coli BL21(DE3) are lysogens of the bacteriophage λ derivative λDE3 (Studier and Moffatt, 1986). In these lysogenic strains, T7 gene 1 (encoding bacteriophage T7 RNA polymerase) is expressed from the lacUV5 promoter, a highly active variant of the E. coli lac promoter. If lac repressor protein (lacI gene product) is bound to the lac operator DNA, then transcription of T7gpl by E. coli RNA polymerase is prevented. However, upon binding to an inducer, such as allolactose, isopropylthiogalactoside (IPTG), etc., the conformationally altered repressor-inducer complex dissociates from operator DNA, allowing RNA transcription to proceed.
In principle, expression of T7 RNA polymerase from lacUV5 promoters is selectively de-repressed upon addition of inducer to bacterial cells (Studier and Moffat, 1986; Rosenberg et al., 1987). However, in practice, the lacUV5 promoter is subject to considerable “leaky” expression—even in the absence of inducer. This leakiness can lead to buildup of toxic gene products and instability of T7 expression vectors in host E. coli strains prior to induction (Studier et al., 1990; Chen, 1994; FIG. 1).
Strategies to Minimize Leaky Expression in T7 Plasmid Cloning Vectors:
One strategy employed to limit “leaky” expression of T7 promoter-dependent target genes is to augment transcriptional control by positioning a lac operator sequence adjacent to plasmid T7 promoters. Transcription from such T7-lac hybrid promoters is largely inhibited when the lac repressor protein occupies the operator (Dubendorff and Studier, 1991).
Unfortunately, the introduction of multi-copy plasmids containing T7-lac promoters into E. coli cells increases the number of lac operator DNA sequences. As a result, endogenous lac repressor protein, which is normally present at about ten copies per cell, is titrated. In the absence of sufficient lac repressor protein, both the lacUV5 promoter controlling T7 RNA polymerase and the T7-lac promoter controlling the expression of the target protein become derepressed.
In order to ensure that sufficient lac repressor is available to maintain repression of T7-lac and lacUV5 promoters, several strategies have been employed to increase lac repressor expression in the host cell. One strategy of increasing lac repressor expression is by incorporating high-expressing alleles of the lacI gene into the T7-lac expression vector (Dubendorff and Studier, 1991; Peranen et al., 1996). Mutant alleles of the lacI promoter have been described which express elevated levels of lac repressor protein. One such allele, lacIq, contains a single point mutation in the −35 promoter element of lacI which increases the amount of lac repressor protein by 10-fold, as compared to wild-type E. coli (Calos, 1978; U.S. Pat. No. 6,569,669 to Raleigh). For low-copy vectors maintained at ˜20 copies/cell, the combination of increased transcriptional activity of the lacIq promoter and increased copy number of the repressor gene is expected to result in a level of lac repressor that is ˜200-fold higher than is normally produced from a single chromosomal copy of wild-type lacI. This increased level of repressor protein is expected to provide for tighter repression of target genes under control of a T7-lac promoter.
However, increasing the expression of the lac repressor protein to minimize background expression can be detrimental to protein production under certain modes of induction. Studies have shown that high levels of lac repressor protein resulting from the lacIq allele reduce the ability to induce protein expression through auto-induction (Blommel et al., 2007 and U.S. Pub. No. 2008/0286749 to Fox et al.).
Thus, a delicate balance between the lac repressor expression level and the number of lac operator sites must be attained for a system to both minimize background expression under uninduced conditions and permit acceptable expression levels upon induction for use with a variety of modes of induction.